Immuno inhibitory pyrazolone compounds

ABSTRACT

Compounds of formula (IA) or (IB) are inhibitors of CD80 and useful in immunomodulation therapy: wherein Ar represents an optionally substituted monocyclic or bicyclic aromatic or heteroaromatic group having from 5 to 10 ring atoms; R 1  and R 2  independently represent H, or C 1 -C 6  alkyl; R 3  represents H; F; CI; Br, —NO 2 ; —CN; C 1 -C 6  alkyl optionally substituted by F or CI; or C 1 -C 6  alkoxy optionally substituted by F; R 4  represents a carboxylic acid group (—COOH) or an ester thereof, or —C(═O)NR 6 R 7 , —NR 7 C(═O)R 6 , —NR 7 C(═O)OR 6 , —NHC(═O)NR 7 R 6  or —NHC(═S)NR 7 R 6  wherein R 6  represents H, or a radical of formula -(Alk) m -Q wherein m is 0 or 1, Alk is an optionally substituted divalent straight or branched C 1 -C 12  alkylene, or C 2 -C 12  alkenylene, or C 2 -C 12  alkynylene radical or a divalent C 3 -C 12  carbocyclic radical, any of which radicals may be interrupted by one or more —O—, —S— or —N(R 8 )— radicals wherein R 8  represents H or C 1 -C 4  alkyl, C 3 -C 4  alkenyl, C 3 -C 4  alkynyl, or C 3 -C 6  cycloalkyl, and Q represents H; —CF 3 ; —OH; —SH; —NR 8 R 8  wherein each R 8  may be the same or different, or form a ring when taken together with the nitrogen to which they are attached; an ester group; or an optionally substituted aryl, aryloxy, cycloalkyl, cycloalkenyl or heterocyclic group; and R 7  represents H or C 1 -C 6  alkyl; or when taken together with the atom or atoms to which they are attached R 6  and R 7  form a monocyclic heterocyclic ring having 5, 6 or 7 ring atoms; and X represents a bond or a divalent radical of formula -(Z) n -(Alk)- or -(Alk)-(Z) n - wherein Z represents —O—, —S— or —NH—, Alk is as defined in relation to R 6  and n is 0 or 1.

This application is a U.S. National Stage application of co-pending PCTapplication PCT/GB2004/004629, filed Nov. 2, 2004, which claims thepriority of Great Britain Patent Application No. 0325644.3, filed Nov.4, 2003. These applications are incorporated herein by reference in itsentirety.

The present invention relates to pyrazolone compounds, to methods fortheir preparation, to compositions containing them, and to methods anduse for clinical treatment of medical conditions which may benefit fromimmunomodulation, e.g. rheumatoid arthritis, multiple sclerosis,diabetes, asthma, transplantation, systemic lupus erythematosis andpsoriasis. More particularly the present invention relates to pyrazolonecompounds, which are CD80 antagonists capable of inhibiting theinteractions between CD80 and CD28.

BACKGROUND TO THE INVENTION

The immune system possesses the ability to control the homeostasisbetween the activation and inactivation of lymphocytes through variousregulatory mechanisms during and after an immune response. Among theseare mechanisms that specifically inhibit and/or turn off an immuneresponse. Thus, when an antigen is presented by MHC molecules to theT-cell receptor, the T-cells become properly activated only in thepresence of additional co-stimulatory signals. In the absence of theseaccessory signals there is no lymphocyte activation and either a stateof functional inactivation termed anergy or tolerance is induced, or theT-cell is specifically deleted by apoptosis.

One such co-stimulatory signal involves interaction of CD80 onspecialised antigen-presenting cells with CD28 on T-cells, and thissignal has been demonstrated to be essential for full T-cell activation.(Lenschow et al. (1996) Annu. Rev. Immunol., 14, 233-258). It wouldtherefore be desirable to provide compounds which inhibit this CD80/CD28interaction.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention there is provided a compound offormula (IA) or (IB) or a pharmaceutically or veterinarily acceptablesalt, hydrate or solvate thereof:

wherein

-   Ar represents an optionally substituted monocyclic or bicyclic    aromatic or heteroaromatic group having from 5 to 10 ring atoms,-   R₁ and R₂ independently represent H, or C₁-C₆ alkyl;-   R₃ represents H; F; Cl; Br; —NO₂; —CN; C₁-C₆ alkyl optionally    substituted by F or Cl; or C₁-C₆ alkoxy optionally substituted by F;-   R₄ represents a carboxylic acid group (—COOH) or an ester thereof,    or —C(═O)NR₆R₇, —NR₇C(═O)R₆, —NR₇C(═O)OR₆, —NHC(═O)NR₇R₆ or    —NHC(═S)NR₇R₆ wherein    -   R₆ represents H, or a radical of formula —(Alk)_(m)—Q wherein        -   m is 0 or 1        -   Alk is an optionally substituted divalent straight or            branched C₁-C₁₂ alkylene, or C₂-C₁₂ alkenylene, or C₂-C₁₂            alkynylene radical or a divalent C₃-C₁₂ carbocyclic radical,            any of which radicals may be interrupted by one or more —O—,            —S— or —N(R₈)— radicals wherein R₈ represents H or C₁-C₄            alkyl, C₃-C₄ alkenyl, C₃-C₄ alkynyl, or C₃-C₆ cycloalkyl,            and        -   Q represents H; —CF₃; —OH; —SH; —NR₈R₈ wherein each R₈ may            be the same or different, or form a ring when taken together            with the nitrogen to which they are attached; an ester            group; or an optionally substituted aryl, aryloxy,            cycloalkyl, cycloalkenyl or heterocyclic group; and    -   R₇ represents H or C₁-C₆ alkyl; or when taken together with the        atom or atoms to which they are attached R₆ and R₇ form a        monocyclic heterocyclic ring having 5, 6 or 7 ring atoms; and-   X represents a bond or a divalent radical of formula —(Z)_(n)-(Alk)-    or -(Alk)-(Z)_(n)- wherein Z represents —O—, —S— or —NH—, Alk is as    defined in relation to R₆ and n is 0 or 1.

Compounds (IA) may exist in the form of tautomers (IA¹):

Hereafter, the compounds (IA) of the invention may be represented andreferred to in either tautomeric form and it is to be understood thatany and all tautomeric forms of structures (IA) and (IB), in particular(IA¹), are included in the invention.

Compounds of general formula (IA) and (IB) are CD80 antagonists. Theyinhibit the interaction between CD80 and CD28 and thus the activation ofT cells, thereby modulating the immune response.

Accordingly the invention also includes:

-   (i) a compound of formula (IA) or (IB) or a pharmaceutically or    veterinarily acceptable salt thereof for use in the treatment of    conditions which benefit from immunomodulation.-   (ii) the use of a compound of formula (IA) or (IB) or a    pharmaceutically or veterinarily acceptable salt thereof in the    manufacture of a medicament for the treatment of conditions which    benefit from immunomodulation.-   (iii) a method of immunomodulation in mammals, including humans,    comprising administration to a mammal in need of such treatment an    immunomodulatory effective dose of a compound of formula (IA) or    (IB) or a pharmaceutically or veterinarily acceptable salt thereof.-   (iv) a pharmaceutical or veterinary composition comprising a    compound of formula (IA) or (IB) or a pharmaceutically or    veterinarily acceptable salt thereof together with a    pharmaceutically or veterinarily acceptable excipient or carrier.    Conditions which Benefit from Immunomodulation Include:-   Acute Disseminated Encephalomyelitis-   Adrenal Insufficiency-   Allergic angiitis and granulomatosis-   Amylodosis-   Ankylosing spondylitis-   Asthma-   Autoimmune Addison's disease-   Autoimmune alopecia-   Autoimmune chronic active hepatitis-   Autoimmune haemolytic anaemia-   Autoimmune Neutrogena-   Autoimmune thrombocytopenic purpura-   Behçet's disease-   Cerebellar degeneration-   Chronic active hepatitis-   Chronic inflammatory demyelinating polyradiculoneuropathy-   Chronic neuropathy with monoclonal gammopathy-   Classic polyarteritis nodosa-   Congenital adrenal hyperplasia-   Cryopathies-   Dermatitis herpetiformis-   Diabetes-   Eaton-Lambert myasthenic syndrome-   Encephalomyelitis-   Epidermolysis bullosa acquisita-   Erythema nodosa-   Gluten-sensitive enteropathy-   Goodpasture's syndrome-   Guillain-Barre syndrome-   Hashimoto's thyroiditis-   Hyperthyroidism-   Idiopathic hemachromatosis-   Idiopathic membranous glomerulonephritis-   Isolated vasculitis of the central nervous system-   Kawasaki's disease-   Minimal change renal disease-   Miscellaneous Vasculitides-   Mixed connective tissue disease-   Multifocal motor neuropathy with conduction block-   Multiple sclerosis-   Myasthenia gravis-   Opsoclonus-myoclonus syndrome-   Pemphigoid-   Pemphigus-   pernicious anaemia-   Polymyositis/dermatomyositis-   Post-infective arthritides-   Primary biliary sclerosis-   Psoriasis-   Reactive arthritides-   Reiter's disease-   Retinopathy-   Rheumatoid arthritis-   Sclerosing Cholangitis-   Sjögren's syndrome-   Stiff-man syndrome-   Subacute thyroiditis-   Systemic lupus erythematosis-   Systemic necrotizing vasculitides-   Systemic sclerosis (scleroderma)-   Takayasu's arteritis-   Temporal arteritis-   Thromboangiitis obliterans-   Type I and type II autoimmune polyglandular syndrome-   Ulcerative colitis-   Uveitis-   Wegener's granulomatosis

As used herein, the term “ester” refers to a group of the form —COOR,wherein R is a radical notionally derived from the alcohol ROH. Examplesof ester groups include the physiologically hydrolysable esters such asthe methyl, ethyl, n- and iso-propyl, n-, sec- and tert-butyl, andbenzyl esters.

As used herein the term “alkylene” refers to a straight or branchedalkyl chain having two unsatisfied valencies, for example —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, —CH(CH₃)CH₂—, —CH(CH₂CH₃)CH₂CH₂CH₂—, and—C(CH₃)₃.

As used herein the term “alkenylene” refers to a straight or branchedalkenyl chain having two unsatisfied valencies, for example—CH═CH—,—CH₂CH═CH—, —C(CH₃)═CH—, and —CH(CH₂CH₃)CH═CHCH₂—.

As used herein the term “alkynylene” refers to a straight or branchedalkynyl chain having two unsatisfied valencies, for example —C≡C—,—CH₂C≡C—, and —CH(CH₂CH₃)C≡CCH₂—.

Unless otherwise specified in the context in which it occurs, the term“substituted” as applied to any moiety herein means substituted with atleast one substituent, for example selected from (C₁-C₆)alkyl,trifluoromethyl, (C₁-C₆)alkoxy (including the special case where a ringis substituted on adjacent ring C atoms by alkylenedioxy such asmethylenedioxy or ethylenedioxy), trifluoromethoxy, (C₁-C₆)alkylthio,phenyl, benzyl, phenoxy, benzyloxy, hydroxy, mercapto, amino, fluoro,chloro, bromo, cyano, nitro, oxo, —COOH, —SO₂OH, —CONH₂, —SO₂NH₂,—COR^(A), —COOR^(A), —SO₂OR^(A), —NHCOR^(A), —NHSO₂R^(A), —CONHR^(A),—SO₂NHR^(A), —NHR^(A), —NR^(A)R^(B), CONR^(A)R^(B) or —SO₂NR^(A)R^(B)wherein R^(A) and R^(B) are independently a (C₁-C₆)alkyl or C₂-C₆ alkoxygroup or a monocyclic carbocyclic or heterocyclic group of from 5-7 ringmembers, or R^(A) and R^(B) form a ring when taken together with thenitrogen to which they are attached. In the case where “substituted”means substituted by phenyl, benzyl, phenoxy, or benzyloxy, the phenylring thereof may itself be substituted with any of the foregoing, exceptphenyl, benzyl, phenoxy, or benzyloxy.

As used herein the term “aryl” refers to a mono-, bi- or tri-cycliccarbocyclic aromatic radical, and to two such radicals covalently linkedto each other, Illustrative of such radicals are phenyl, biphenyl andnapthyl.

As used herein the unqualified term “carbocyclyl” or “carbocyclic”includes aryl, cycloalkyl and cycloalkenyl and refers to a ring system(monocyclic, bicyclic, tricyclic or bridged) whose ring atoms are allcarbon.

As used herein the unqualified term “cycloalkyl” refers to a carbocyclicring system which contains only single bonds between ring carbons.

As used herein the unqualified term “cycloalkenyl” refers to acarbocyclic ring system which contains at least one double bond betweena pair of ring carbons.

As used herein the term “heteroaryl” refers to a mono-, bi- ortri-cyclic aromatic radical containing one or more heteroatoms selectedfrom S, N and O. Illustrative of such radicals are thienyl, benzthienyl,furyl, benzfuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl,benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl,benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl,benztriazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl.

As used herein the unqualified term “heterocyclyl” or “heterocyclic”includes “heteroaryl” as defined above, and in particular means a mono-,bi- or tri-cyclic or bridged non-aromatic radical containing one or moreheteroatoms selected from S, N and O, and to groups consisting of amonocyclic non-aromatic radical containing one or more such heteroatomswhich is covalently linked to another such radical or to a monocycliccarbocyclic radical. Illustrative of such radicals are pyrrolyl,furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl,thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl,pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl,benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl,ethylenedioxyphenyl, maleimido and succinimido groups.

Some compounds of the invention contain one or more chiral centresbecause of the presence of asymmetric carbon atoms. The presence ofasymmetric carbon atoms gives rise to stereoisomers or diastereoisomerswith R or S stereochemistry at each chiral centre. The inventionincludes all such stereoisomers and diastereoisomers and mixturesthereof.

Salts of salt forming compounds of the invention include physiologicallyacceptable acid addition salts for example hydrochlorides,hydrobromides, sulphates, methane sulphonates, p-toluenesulphonates,phosphates, acetates, citrates, succinates, lactates, tartrates,fumarates and maleates; and base addition salts, for example sodium,potassium, magnesium, and calcium salts.

Methods

Compounds of the invention of formula (IA) wherein R₁ is hydrogen andwherein R₄ represents an amide group —C(═O)NR₆R₇ may be prepared byreaction of the appropriate amine HNR₆R₇ with a compound of formula (II)to amidate the carboxylic acid group:

the symbols Ar, R₃, X, R₆ and R₇ being as defined in relation to formula(I) above.

Compounds (II) (ie compounds (IA) of the invention wherein R₁ ishydrogen and R₄ is a carboxylic acid group) may be prepared by reactionof a compound of formula (III) with a hydrazine of formula (IV):

This reaction may result in the preparation of a mixture of the positionisomers (IIA) and (IIB):

from which the desired isomer (IIA) may be separated.

Compounds (IA) wherein R₁ is hydrogen and R₄ is an amide (—C(═O)NR₆R₇)or ester group may also be prepared from intermediate (III) by reactionwith the appropriate hydrazine (IVA)

wherein R₄ is the amide or ester group. Again the reaction may result ina mixture of the ester analogues of the carboxylic acids (IIA) and(IIB), from which the desired ester isomer (I) may be separated.Alternatively, the carboxylic acid compound (II) may simply beesterified, or amidated (the latter being a route referred to above).

Compounds (IA) wherein R₁ is hydrogen and R₄ is a “reverse amide” group—NR₇C(═O)R₆ may be prepared by Curtius rearrangement (see Ninomiya, K.;Shioiri, T.; Yamada, S. Tetrahedron (1974), 30(14), 2151-7) of thecarboxylic acid (II) to the isocyanate (V)

followed by hydrolysis of the isocyanate group to an amino group andacylation of the amino group with, for example, the acid chlorideCl—C(═O)R₆. In cases where R₇ is not hydrogen, the R₇ substituent may beintroduced after the isocyanate reduction step or after the acylationstep.

Compounds (IA) wherein R₁ is hydrogen and R₄ is a urea group—NHC(═O)NHR₆ or thiourea group —NHC(═S)NHR₆ may also be prepared fromthe isocyanate (V) or the corresponding isothiocyanate by reaction withthe appropriate amine H₂NR₆

Compounds (I) wherein R₄ is a carbamate group —NR₇C(═O)OR₆ may beprepared by the reaction of the isocyanate with an appropriate alcoholR₆OH.

Compounds (IA) and (IB) wherein R₁ and R₂ are C₁-C₆ alkyl may beprepared by alkylation of the corresponding compound (IA) wherein R₁ ishydrogen.

Further details of the synthetic methods for the preparation ofcompounds (I) of the invention, and intermediates such as (III), may befound in the examples herein.

In the compounds of the invention:

R₁ and R₂ independently represent hydrogen or C₁-C₆ alkyl, such asmethyl, ethyl, n- or iso-propyl, n-, sec- or tert-butyl.

R₄ represents a carboxylic acid group (—COOH) or an ester thereof, or—C(═O)NR₆R₇, —NR₇C(═O)R₆, —NR₇C(═O)OR₆ or —NHC(═O)NHR⁶, all as definedabove.

-   -   When R₄ is an ester group, examples include those of formula        —COOR wherein R is methyl, ethyl n- or iso-propyl, n-, sec- or        tert-butyl, or benzyl ester.    -   R₆, when present, represents H, or a radical of formula        -(Alk)_(m)-Q wherein m, Alk and Q being as defined above. When m        is 1, Alk may be, for example a straight or branched C₁-C₆        alkylene radical, such as —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, and        —CH₂CH(CH₃)CH₂—. Alk may also be, for example, a divalent        cyclopropylene, cyclopentylene or cyclohexylene radical. The        radical Alk may be optionally substituted by, for example, OH,        oxo, CF₃, methoxy or ethoxy. The radical Alk may optionally        contain a hetero atom, for example in the form of an ether,        thioether or amino linkage.    -   The group Q may represent, for example, hydrogen; —NR₈R₈ wherein        each R₈ may be the same or different and selected from hydrogen,        methyl, ethyl, n- or isopropyl or tert-butyl; an ester group for        example a methyl, ethyl or benzyl ester; or an optionally        substituted aryl, aryloxy, cycloalkyl, cycloalkenyl or        heterocyclic group, for example phenyl, phenoxy, cyclopentyl,        cyclohexyl, furyl, thienyl, piperidyl, or piperazinyl group.    -   R₇ when present represents H or C₁-C₆ alkyl, for example methyl,        ethyl n- or iso-propyl, n-, sec- or tert-butyl; or when taken        together with the atom or atoms to which they are attached R₆        and R₇ form a monocyclic heterocyclic ring having 5, 6 or 7 ring        atoms;

Ar may be, for example, optionally substituted phenyl, 2-, 3-, or4-pyridyl, 2-, or 3-furyl, 2-, or 3-thienyl, benzfur-2-yl, orbenzthien-2-yl. Optional substituents in Ar include, for example F, Cl,methyl, methoxy, or methylenedioxy. Currently it is preferred that Ar is3-fluorophenyl and 2-, or 3-furyl,

R₃ may be, for example, H, F, Cl, methyl, methoxy, or methylenedioxy.Currently it is preferred that R₃ is H;

X may be, for example a bond, or a —CH₂— or —CH₂CH₂— radical. A bond ispresently preferred.

A specific preferred subset of compounds of the invention has formula(IC):

wherein Ar, R₆ and R₇ are as specified above. In this subset, theradical —C(═O)NR₆R₇ may be in the 4-position of the phenyl ring. Thissubset includes in particular, compounds wherein R₇ is hydrogen and R₆is -AlkNR₈R₈ wherein the R₈ groups are is as defined above.

Specific compounds of the invention include those of the Examplesherein.

As mentioned above, the invention includes pharmaceutical or veterinarycomposition comprising a compound of formula (I) or a pharmaceuticallyor veterinarily acceptable salt thereof together with a pharmaceuticallyor veterinarily acceptable excipient or carrier. In such compositions,it will be understood that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination and the cause and severity of the particulardisease undergoing therapy. Optimum dose levels and frequency of dosingwill be determined by clinical trial.

The compounds with which the invention is concerned may be prepared foradministration by any route consistent with their pharmacokineticproperties. The orally administrable compositions may be in the form oftablets, capsules, powders, granules, lozenges, liquid or gelpreparations, such as oral, topical, or sterile parenteral solutions orsuspensions. Tablets and capsules for oral administration may be in unitdose presentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricant, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants for example potato starch, or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavouring or colouring agents.

For topical application to the skin, the drug may be made up into acream, lotion or ointment. Cream or ointment formulations which may beused for the drug are conventional formulations well known in the art,for example as described in standard textbooks of pharmaceutics such asthe British Pharmacopoeia.

For topical application to the eye, the drug may be made up into asolution or suspension in a suitable sterile aqueous or non aqueousvehicle. Additives, for instance buffers such as sodium metabisulphiteor disodium edeate; preservatives including bactericidal and fungicidalagents such as phenyl mercuric acetate or nitrate, benzalkonium chlorideor chlorhexidine, and thickening agents such as hypromellose may also beincluded.

The active ingredient may also be administered parenterally in a sterilemedium. Depending on the vehicle and concentration used, the drug caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as a local anaesthetic, preservative and buffering agentscan be dissolved in the vehicle.

Embodiments of the Invention are Described in the Following Non-limitingExamples

The following abbreviations are used in the experimental descriptions:

-   DMF Dimethyl formamide-   DMA Dimethyl acetamide-   DMSO Dimethyl sulphoxide-   THF Tetrahydrofuran-   HBTU O-Benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HPLC High performance liquid chromatography-   LCMS Liquid chromatography mass spectrum-   NMR Nuclear magnetic resonance spectroscopy

EXAMPLE 14-(5-Oxo-3-pyrazin-2-yl-2,5-dihydro-pyrazol-1-yl)-N-(1,2,2,6,6-pentamethyl-piperidin-4-yl)-benzamide

Step 1: Preparation of 4-(N′-tert-Butoxycarbonyl-hydrazino)-benzoic acid

To a deep red solution of p-hydrazinobenzoic acid (32.86 mmol, 5.00 g)in 2M NaOH (100 ml) and THF (70 ml) was added di-tertbutyl dicarbonate(2.0 eq, 65.72 mmol, 14.34 g). This was stirred at rt over a weekend.Another aliquot of di-tertbutyl dicarbonate (3.5 g) was added to thereaction mixture, and this stirred at room temp for a further 24 hours.A further 0.5 eq p-hydrazinobenzoic acid added to the reaction mixture.This was then stirred for a further 24 hours. 10% citric acid solutionwas added until a pH of 4 was obtained. The product was then extractedwith ethyl acetate, the organics washed with brine, and then dried overNa₂SO₄. Concentration in vacuo afforded a pale yellow oil, containingresidual tert-BuOH. Hexane was added to the mixture. Precipitation wasobserved and the solids were collected by filtration, washed with hexaneand dried under vacuum at 35° C. An orange powder was obtained (8.40 g).

Step 2: Preparation ofN′-[4-(1,2,2,6,6-Pentamethyl-piperidin-4-ylcarbamoyl)-phenyl]-hydrazinecarboxylicacid tert-butyl ester

To a solution of 4-(N′-tert-Butoxycarbonyl-hydrazino)-benzoic acid(4.452 g) in DMA (30 ml) was added disopropyl ethyl amine (6.16 ml) andHBTU (6.69 g) with stirring at room temp.4-Amino-1,2,2,6,6-pentamethylpiperidine (3 g) was then added to thestirred solution. Stirring continued at room temp for 4 hrs.

The solution was partitioned between ethyl acetate (6×20 ml) and water.The organic fraction was taken, washed with brine, dried over MgSO₄ andthe solvent removed under vacuum. This revealed a deep orange oil (6.2g, 86%).

Step 3: Preparation of4-Hydrazino-N-(1,2,2,6,6-pentamethyl-piperidin-4-yl)-benzamide

N′-[4-(1,2,2,6,6-Pentamethyl-piperidin-4-ylcarbamoyl)-phenyl]-hydrazinecarboxylicacid tert-butyl ester was dissolved in 4M HCl in dioxane (30 ml) andleft to stir for 1-2 hrs. The solution went quickly from a deep orangecolour to a bright orange colour. After 1 hr reaction time, the solventwas removed under vacuum to reveal an orange oil that under a vacuum forseveral minutes crystalised out to give orange crystals in a stickyorange oil.

Yield: 4.68 g, 87%.

Step 4: Preparation of 3-Oxo-3-pyrazin-2-yl-propionic acid, ethyl ester

To a stirred solution of diethyl malonate (1.25 ml, 1.32 g) in THF (20ml) under an atmosphere of nitrogen at −60° C. was added nbutyl lithium(2.5 M, 9 ml) dropwise over 15 min, keeping the temperature constant.The reaction became a cloudy white colour from the formation of thedi-lithium salt then became yellowish. After 10 min, the reaction wascooled to −78° C., when a solution of pyrazine-2-carbonyl chloride (0.5g, dark purple solution in THF (10 ml)) was added dropwise over 15 min.The reaction then warmed to −45° C. and stirred for 1 h. The reactionwas then poured into 1 M HCl solution (35 ml) with stirring. This wastransferred to a separating funnel, and extracted with CH₂Cl₂ (2×150ml). The combined organic layers were then washed with sat. NaCO₃ soln(1×30 ml) and then dried over MgSO₄, filtered and concentrated in vacuoto give the product as a brown oil. Yield 0.65 g, 45%.

Step 5: Preparation of4-(5-Oxo-3-pyrazin-2-yl-2,5-dihydro-pyrazol-1-yl)-N-(1,2,2,6,6-pentamethyl-piperidin-4-yl)-benzamide

3-Oxo-3-pyrazin-2-yl-propionic acid, ethyl ester (78 mg) and4-hydrazino-N-(1,2, 2,6,6-pentamethyl-piperidin-4-yl)-benzamide (141 mg)were dissolved in ethanol (3.5 ml) and acetic acid (0.5 ml) and thesolution stirred at 65° C. for 2 h. Concentration in vacuo andpurification by HPLC gave the expected product. LC/MS: main peak isproduct (m/z: 434)

EXAMPLE 24-(3-Isoxazol-5-yl-5-oxo-2,5-dihydro-pyrazol-1-yl)-N-(1,2,2,6,6-pentamethyl-piperidin-4-yl)-benzamide

Step 1: Preparation of 3-Isoxazol-5-yl-3-oxo-propionic acid ethyl ester

To a stirred solution of diethyl malonate (2.6 ml, 2.74 g) in THF (40ml) under an atmosphere of nitrogen at −60° C. was added nbutyl lithium(2.5 M, 18.9 ml) dropwise over 15 mins, keeping the temperatureconstant. The reaction became a cloudy white colour from the formationof the di-lithium salt then became yellowish. After 10 mins, thereaction was cooled to −78° C., when a solution of Isoxazole-5-carbonylchloride (1 g) in THF (10 ml) was added dropwise over 15 mins. Thereaction then warmed to −45° C. and stirred for 1 h, the solution hadgone pale brown. The reaction was then poured into 1 M HCl solution (50ml) with stirring and extracted with CH₂Cl₂ (2×150 ml). The combinedorganic layers were washed with sat. NaCO₃ soln (1×30 ml), dried overMgSO₄, filtered and concentrated in vacuo to give the product as a paleoil which was used as such in the next step. Yield 1.1 g, 35.1%.

Step 2: Preparation of4-(3-Isoxazol-5-yl-5-oxo-2,5-dihydro-pyrazol-1-yl)-N-(1,2,2,6,6-pentamethyl-piperidin-4-yl)-benzamide

3-Isoxazol-5-yl-3-oxo-propionic acid ethyl ester (49 mg) and4-Hydrazino-N-(1,2, 2,6,6-pentamethyl-piperidin-4-yl)-benzamide (93 mg)were dissolved into ethanol (2.5 ml) and acetic acid (0.3 ml) and thesolution stirred at 65° C. for 2 h. Concentration in vacuo andpurification by HPLC gave the expected product. LC/MS: main peak isproduct (m/z: 424.3)

The compound of Example 2 had activity rating * in the HTRF assaydescribed below.

EXAMPLE 34-(3-Furan-2-yl-4-methyl-5-oxo-2,5-dihydro-pyrazol-1-yl)-N-(1,2,2,6,6-pentamethyl-piperidin-4-yl)-benzamide

Step 1: Preparation of 3-furan-2-yl-2-methyl-3-oxo-propionic acid ethylester

In a flask were placed ethyl 3-(2-furyl)-3-oxopropanoate (0.5 g),iodomethane (0.14 ml), finely ground potassium carbonate (0.75 g) andacetone (5 ml). The mixture was stirred at reflux for 2 h. A furtherportion of iodomethane (0.9 mmol, 0.056 ml) and potassium carbonate (0.9mmol, 0.12 g) were added and the mixture stirred at reflux for 1 h. Thereaction mixture was filtered and concentrated under vacuum. Yield (0.5g, 93%).

Preparation of4-(3-Furan-2-yl-4-methyl-5-oxo-2,5-dihydro-pyrazol-1-yl)-N-(1,2,2,6,6-pentamethyl-piperidin-4-yl)-benzamide

3-furan-2-yl-2-methyl-3-oxo-propionic acid ethyl ester (49 mg) and4-Hydrazino-N-(1,2,2,6,6-pentamethyl-piperidin-4-yl)-benzamide (85 mg)in 0.5 ml of acetic acid were heated up at 65° C. for 2 h. Concentrationin vacuo and purification by HPLC gave the expected product. MH+=437.3

The compound of Example 3 had activity rating * in the HTRF assaydescribed below.

EXAMPLE 44-(3-Furan-2-yl-4,4-dimethyl-5-oxo-4,5-dihydro-pyrazol-1-yl)-N-(1,2,2,6,6-pentamethyl-piperidin-4-yl)-benzamide

Step 1: Preparation of 3-Furan-2-yl-2,2-dimethyl-3-oxo-propionic acidethyl ester

3-furan-2-yl-2-methyl-3-oxo-propionic acid ethyl ester (49 mg) was addedsodium ethoxide (0.22 ml) and 0.5 ml ethanol. A solid precipitates. Thesolution was stirred at room temperature for 1 h, iodomethane was addedand the suspension stirred at room temperature for 3 h. Concentration invacuo and purification by HPLC gave the expected product.

Step 2: Preparation of4-(3-Furan-2-yl-4,4-dimethyl-5-oxo-4,5-dihydro-pyrazol-1-yl)-N-(1,2,2,6,6-pentamethyl-piperidin-4-yl)-benzamide

3-Furan-2-yl-2,2-dimethyl-3-oxo-propionic acid ethyl ester (53 mg) and4-Hydrazino-N-(1,2,2,6,6-pentamethyl-piperidin-4-yl)-benzamide (85 mg)in 0.5 ml of acetic acid were heated up at 65° C. for 2 h. Concentrationin vacuo and purification by HPLC gave the expected product (MH+: 451.4)

ADDITITIONAL EXAMPLES

Further examples of compounds of the invention were synthesised bymethods analogous to those of Examples 1-2 above. The structures of thesynthesised compounds are shown in the following Table, together withtheir activity ratings in the HTRF assay described below.

TABLE 1

Activity Example Z W R MH+ Rating  5 Ph H CH₂CH₂CH₂N(Me)₂ 365.2 **  6 PhH

482.1 *  7 Ph H

433.5 **  8 Ph H

405.5 ***  9 Ph H

419.5 ** 10 2-Pyridyl H CH₂CH₂CH₂N(Me)₂ 366.5 *** 11 2-furyl HCH₂CH₂CH₂N(Me)₂ 355.4 *** 12 3-Pyridyl H CH₂CH₂CH₂N(Me)₂ 366.5 ** 132-Pyridyl H

420.5 ** 14 2-furyl H

409.5 *** 15 3-Pyridyl H

420.5 ** 16 2-Pyridyl H

434.6 *** 17 3-furyl H CH₂CH₂CH₂N(Me)₂ 355.5 ** 18 3-furyl H

409.5 *** 19 2-furyl H

423.6 *** 20 3-furyl H

423.6 ** 21 2-furyl H

395.6 ** 22 3-furyl H

395.6 ** 23 3-Pyridyl H

434.6 ** 24 2-Pyridyl H

406.5 *** 25 3-Pyridyl H

406.6 ** 26 3F-phenyl H CH₂CH₂CH₂N(Me)₂ 383.5 *** 27 3F-phenyl H

437.6 ** 28 3F-phenyl H

451.6 *** 29 2-pyrazinyl H

407.3 ** 30 2-pyrazinyl H CH₂CH₂CH₂N(Me)₂ 367.3 *** 31 2-benzofuranyl H

445.3 ** 32 2-benzofuranyl H CH₂CH₂CH₂N(Me)₂ 405.3 ** 33 2-benzofuranylH

459.4 ** 34 2-benzofuranyl H

473.4 ** 35 5-isoxazolyl H

424.3 *Assay ProtocolsThe Use of Biacore Biomolecular Interaction Analysis

Biotinylated human CD80 (hCD80-BT) is a recombinant soluble form of amembrane bound receptor molecule (CD80) which binds to CD28 to initiateT cell activation. The interaction between CD80 and CD28 has beenextensively investigated (Collins et al., 2002). Biotinlyated humanHLA-A2-tax is the recombinant soluble form of a membrane bound receptormolecule that has been used in this example as a control protein, and isnot expected to interact with the compounds.

The BIAcore S51™ system was used for screening the compounds of Examples1-4 above. A series S sensor chip CM5 was docked onto the BIAcore S51™.Streptavidin was coupled to the carboxymethyl surface using standardamine coupling. The chip surface was activated with 0.2M EDC/0.05M NHS,followed by binding of streptavidin (0.25 mg/ml in 10 mM sodium acetatepH 5.0) and saturation of unoccupied sites with 1 M ethylenediamine.

The BIAcore S51 sensor chip has two separate sensor spots forimmobilisation of proteins. hCD80-BT was immobilised on thestreptavidin-coated surface of one sensor spot until a response ofapproximately 3000 RU was observed. A protein to control fornon-specific binding of the compound was immobilised on a second sensorspot. The control protein used for these experiments was a biotinylated,soluble form of the human HLA protein.

Dilution series of compounds (1000 nM-0.05 nM) were prepared in runningbuffer (10 mM, pH 7.4, 150 mM NaCl, 0.005% P20; 5% DMSO).

BIAcore S51™ was run at a flow rate of 30 μl/min using running buffer.Compounds and DMSO standard solutions for correction of data for solventeffects were injected. Data were recorded automatically and wereanalysed using BIAcore S51 Evaluation software.

The interaction between CD80 and the endogenous protein ligand (CD28) ishighly specific, but relatively weak, with a K_(D) of 4750 nM, and anoff-rate of greater than 0.2 s⁻¹. The compounds of Examples 7, 11 &18-21 have greater affinity and longer residence times on CD80 thanCD28, having K_(D)s of less than 100 nM, and off-rates of 2×10⁻²,indicating that the pyrazolones will be able to compete effectively withthe endogenous ligand. The pyrazolones showed no detectable interactionwith the control protein.

References

Collins A V et al. (2002) Immunity 17, 201-210 “The interactionproperties of costimulatory molecules revisited”

Inhibition of Production of Interleukin-2 (IL-2) by Human Jurkat TCells.

Method

Human Raji cells were dispensed at a concentration of 2×10⁵ cells perwell in RPMI-1640 medium supplemented with 10% fetal calf serum, 1%penicillin/streptomycin, 1% glutamine (RPMI medium) in a 96-well roundbottom microtitre plate. Compounds under investigation (dissolved in100% DMSO) were diluted to eight-fold the desired final concentration inRPMI medium and added to the required final concentration for a totalvolume of 200 μl per well. After 20 minutes incubation at 37° C., JurkatT cells were added at a concentration of 2×10⁵ cells per well.Monoclonal antibody to CD3 (UCHT1, R&D Systems) was added to thecultures at a final concentration of 1 μg per ml, and where indicated,monoclonal antibody to CD28 (CD28.2, BD-Pharmingen) was also added at aconcentration of 2.5 μg per ml. Cells were cultured at 37° C. for 5hours, after which the plates were centrifuged and the supernatantsharvested for IL-2 ELISA assay using the IL-2 Eli-pair kit (DIACLONEResearch, Besancon, France) according to the manufacturers instructions.

By way of example, the compound of Example 26 gave 52% inhibition at 30μM.

Homogenous Time Resolved Fluorescence Assay

The examples described above were tested in a cell free Homogenous TimeResolved Fluorescence (HTRF) assay to determine their activity asinhibitors of the CD80-CD28 interaction.

In the assay, europium and allophycocyanin (APC) are associated withCD28 and CD80 indirectly (through antibody linkers) to form a complex,which brings the europium and APC into close proximity to generate asignal. The complex comprises the following six proteins: fluorescentlabel 1, linker antibody 1, CD28 fusion protein, CD80 fusion protein,linker antibody 2, and fluorescent label 2. The table below describesthese reagents in greater detail.

Fluorescent label 1 Anti-Rabbit IgG labelled with Europium (1 μg/ml)Linker antibody 1 Rabbit IgG specific for mouse Fc fragment (3 μg/ml)CD28 fusion protein CD28 - mouse Fc fragment fusion protein (0.48 μg/ml)CD80 fusion CD80 mouse Fab fragment (C215) fusion protein protein (1.9μg/ml) Linker antibody 2 GαMκ-biotin: biotinylated goat IgG specific formouse kappa chain (2 μg/ml) Fluorescent label 2 SA-APC: streptavidinlabelled allophycocyanin (8 μg/ml)

On formation of the complex, europium and APC are brought into proximityand a signal is generated.

Non-specific interaction was measured by substituting a mouse Fabfragment (C215) for the CD80 mouse Fab fragment fusion protein (1.9μg/ml). The assay was carried out in black 384 well plates in a finalvolume of 30 μl. Assay buffer: 50 mM Tris-HCl, 150 mM NaCl pH7.8,containing 0.1% BSA (w/v) added just prior to use.

Compounds were added to the above reagents in a concentration seriesranging between 100 μM-1.7 nM. The reaction was incubated for 4 hours atroom temperature. Dual measurements were made using a Wallac Victor 1420Multilabel Counter. First measurement: excitation 340 nm, emission 665nm, delay 50 μs, window time 200 μs. second measurement: excitation 340nm, emission 615 nm, delay 50 μs, window time 200 μs. Counts wereautomatically corrected for fluorescence crossover, quenching andbackground. The EC50 activities of compounds tested are recorded as:EC50: *=>10 μM, **=1-10 μM, ***=<1 μM.

1. A compound of formula (IA) or (1B) or a pharmaceutically orveterinarily acceptable salt thereof:

wherein Ar represents an optionally substituted monocyclic or bicyclicaromatic or heteroaromatic group having from 5 to 10 ring atoms, R₁ andR₂ independently represent H, or C₁-C₆ alkyl; R₃ represents H; F; Cl;Br; —NO₂; —CN; C₁-C₆ alkyl optionally substituted by F or Cl; or C₁-C₆alkoxy optionally substituted by F; R₄ represents a carboxylic acidgroup (—COOH) or an ester thereof, or —C(═O)NR₆R₇, —NR₇C(═O)R₆,—NR₇C(═O)OR₆, —NHC(═S)NR₇R₆ or —NHC(═S)NR₇R₆ wherein R₆ represents H, ora radical of formula —(Alk)_(m)-Q wherein m is 0 or 1 Alk is anoptionally substituted divalent straight or branched C₁-C₁₂ alkylene, orC₂-C₁₂ alkenylene, or C₂-C₁₂ alkynylene radical or a divalent C₃-C₁₂carbocyclic radical, any of which radicals may be interrupted by one ormore —O—, —S—or —N(R₈)— radicals wherein R₈ represents H or C₁-C₄alkyl,C₃-C₄ alkenyl, C₃-C₄ alkynyl, or C₃-C₆ cycloalkyl, and Q represents H;—CF₃; —OH; —SH; —NR₈R₈ wherein R₈ is defined as above, and each R₈ maybe the same or different; an ester group; or an optionally substitutedaryl, aryloxy, cycloalkyl, or cycloalkenyl; and R₇ represents H or C₁-C₆alkyl; and X represents a bond or a divalent radical of formula—(Z)_(n)-(Alk)- or -(Alk)-(Z)_(n)- wherein Z represents —O—, —S— or—NH—, Alk is as defined in relation to R₆ and n is 0 or
 1. 2. A compoundas claimed in claim 1 wherein R₁ in compounds (IA) and each of R₁ and R₂in compounds (IB) is other than hydrogen.
 3. A compound as claimed inclaim 1 wherein R₄ represents a carboxylic acid group (—COOH) or anester group of formula —COOR wherein R is methyl, ethyl n- oriso-propyl, n-, sec- or tert-butyl, or benzyl.
 4. A compound as claimedin claim 1 wherein R₆ represents a radical of formula -(Alk)_(m)-Qwherein m is 1, Alk is —CH₂—, CH₂CH₂—, —CH₂CH₂CH₂—, or —CH₂CH(CH₃)CH₂—,or a divalent cyclopropylene, cyclopentylene or cyclohexylene radical,optionally substituted by OH, oxo, CF₃, methoxy or ethoxy, and Qrepresents hydrogen; —NR₈R₈ wherein each R₈ may be the same or differentand selected from hydrogen, methyl, ethyl, n- or isopropyl ortert-butyl; a methyl, ethyl or benzyl ester; or an optionallysubstituted phenyl, phenoxy, cyclopentyl, or cyclohexyl group.
 5. Acompound as claimed in claim 1 wherein R₇ represents hydrogen, methyl,ethyl, n- or iso-propyl, n-, sec- or tert-butyl.
 6. A compound asclaimed in claim 1 wherein Ar is optionally substituted phenyl, 2-, 3-,or 4-pyridyl, 2-, or 3-furyl, 2-, or 3-thienyl, benzfur-2-yl, orbenzthien-2-yl.
 7. A compound as claimed in claim 1 wherein Ar issubstituted by F, Cl, methyl, methoxy, or methylenedioxy.
 8. A compoundas claimed in claim 1 wherein Ar is 3-fluorophenyl, or 2- or 3-furyl. 9.A compound as claimed in claim 1 wherein R₃ is H, F, Cl, methyl,methoxy, or methylenedioxy.
 10. A compound as claimed in claim 1 whereinX is a bond, or a —CH₂or —CH₂CH₂— radical.
 11. A compound as claimed inclaim 1 which is of formula (IC) or a pharmaceutically or veterinarilyacceptable salt thereof:


12. A compound as claimed in claim 11 wherein the radical —C(═O)NR₆R₇ isin the 4-position of the phenyl ring.
 13. A compound as claimed in claim11 wherein R₇ is hydrogen and R₆ is -AlkNR₈R₈ wherein the R₈ representsH or C₁-C₄ alkyl, C₃-C₄ alkenyl, C₃-C₄ alkynyl, or C₃-C6 cycloalkyl. 14.A pharmaceutical or veterinary composition comprising a compound asclaimed in claim 1 together with a pharmaceutically or veterinarilyacceptable excipient or carrier.
 15. A composition according to claim 14wherein the compound is in an amount effective for treatment ofconditions which benefit from immunomodulation.
 16. A medicament for thetreatment of conditions which benefit from immunomodulation comprisingan effective amount of the compound according to claim 1 together with apharmaceutically or veterinarily acceptable excipient or carrier;wherein the conditions are selected from rheumatoid arthritis,psoriasis, multiple sclerosis, and diabetes.